It was extensively demonstrated in the last decades that astrocytes are not passive elements, but actively participate in the neuronal signal processes. For the optimal experimental approach of astrocyte-neuron communication, selective activation of the astrocytes is needed. A recent possibility for this aim is the optogenetics, where certain cell lines express light-activated ion channels and transporters. In the present project we aimed to provide evidence that astrocytic activation by optogenetics is possible in samples from the brainstem.
We used coronal midbrain slices from specific mouse strains where astrocytes expressed either tdTomato fluorescent protein or channelrhodopsin-2 (ChR2) light-activated cationic channel in an astrocytic marker- (GFAP-)dependent way. Recordings were performed in the pedunculopontine nucleus (PPN). The activation of the astrocytes was visualized with calcium imaging using Oregon Green BAPTA I AM or Rhod2 fluorescent dyes. Astrocytes and neurons were also investigated with patch clamp technique in whole cell configuration.
Optogenetic activation of astrocytes was achieved by illumination of the sample with 470 nm light and parallel calcium imaging was performed. The area of the calcium waves recorded from astrocytes was significantly increased during and after illumination (15.5 ± 3.6 %*s before and 93.5 ± 30.3 %*s during photostimulation; p = 0.012; n = 16). Voltage-clamp experiments on astrocytes from the same sample showed that illumination of astrocytes resulted 65.7 ± 2.6 pA inward current (on -80 mV holding potential; n = 6). Neurons in the same samples also responded to activation of astrocytes. Current-clamp experiments on neurons of the PPN revealed that optogenetic activation of astrocytes depolarized the neurons with 5.07 ± 1.4 mV and increased their firing rate with 1.42 ± 0.54 Hz (n = 9).
Our present data indicate that optogenetic activation of astrocytes is possible with our experimental arrangement. Astrocytic activation can massively influence excitability of the surrounding neurons; therefore, this method can be used as a powerful tool to investigate astrocytic contribution to neuronal excitability.